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PubMed1998

P450 isoforms in a murine macrophage cell line, RAW264.7, and changes in the levels of P450 isoforms by treatment of cells with lipopolysaccharide and interferon-gamma.

The presence of P450 in a murine macrophage cell line, RAW264.7, was investigated to clarify the biological role and regulation of P450. Microsomes of RAW264.7 cells were isolated and subjected to immunoblotting with anti-rat CYP2A1, 2B1, and 4A2 antibodies. The microsomes gave staining bands with all these antibodies, suggesting the presence of mouse Cyp2a, 2b, and 4a isoforms in RAW264.7. RAW264. 7 cells were treated with typical inducers of P450 (phenobarbital, clofibrate, beta-naphthoflavone and 3-methylcholanthrene). None of these chemicals induced these P450s. Stimulation of RAW264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (INF-gamma) which increase inducible nitric oxide synthase (iNOS) and cytokines in cells decreased Cyp4a protein but not Cyp2a and 2b proteins. To identify P450 isoforms in RAW264.7, we used polymerase chain reaction (PCR) primers for mouse Cyp2a4, 2a12, 2b9/10, 4a10, and 4a12. Total RNA was isolated from these cells and converted to cDNA by reverse transcriptase. PCR was done with these primers and the amplified nucleotides were analyzed by a DNA sequencer. Only Cyp2b9/10 and 4a12 primers gave clear bands, although all primers gave clear bands from liver total RNA. Nucleotide sequences of these products amplified by PCR were identical with Cyp2b9 and 4a12. These findings indicate that Cyp2b9 and 4a12 were present in a macrophage cell line, RAW264.7, and the regulation of P450 by inducers and cytokine differed from that in liver.

Animals↗

PubMed1991

Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: a model for adrenal growth regulation.

Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cells. Northern blot analysis of total RNA from whole human fetal adrenal glands revealed a characteristic 7-kilobase bFGF mRNA, indicating that the fetal adrenal bFGF bioactivity was most likely due to local synthesis. Slot blot and ribonuclease protection analysis showed that bFGF mRNA was present in very low amounts in total RNA from primary cultures of unstimulated human fetal adrenal cells but was increased 2- to 3-fold in cells exposed to 10 nM ACTH-(1-24) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate for 24 hr. bFGF mRNA was localized to adrenocortical cells and not fibroblasts by in situ hybridization. bFGF mRNA was barely detectable in unstimulated cells, whereas it was markedly increased in cells exposed to either ACTH or 8-bromoadenosine 3',5'-cyclic monophosphate. These data support our hypothesis that the regulation of human fetal adrenal growth by ACTH at midgestation may be mediated by the stimulation of local growth factor production, and we suggest that bFGF may play a major role in this process.

Adrenal Glands↗

PubMed2002

Fatty acid and lipid biosynthetic genes are expressed at constant molar ratios but different absolute levels during embryogenesis.

In plants, fatty acid and complex lipid synthesis requires the correct spatial and temporal activity of many gene products. Quantitative northern analysis showed that mRNA for the biotin carboxylase subunit of heteromeric acetyl-coenzyme A carboxylase, fatty acid synthase components (3-oxoacyl-acyl carrier protein [ACP] reductase, enoyl-ACP reductase, and acyl-ACP thioesterase), and stearoyl-ACP desaturase accumulate in a coordinate manner during Brassica napus embryogenesis. The mRNAs were present in a constant molar stoichiometric ratio. Transcript abundance of mRNAs for the catalytic proteins was found to be similar, whereas the number of ACP transcripts was approximately 7-fold higher. The peak of mRNA accumulation of all products was between 20 and 29 d after flowering; by 42 d after flowering, the steady-state levels of all transcripts fell to about 5% of their peak levels, which suggests that the mRNAs have similar stability and kinetics of synthesis. Biotin carboxylase was found to accumulate to a maximum of 59 fmol mg(-1) total RNA in embryos, which is in general agreement with the value of 170 fmol mg(-1) determined for Arabidopsis siliques (J.S. Ke, T.N. Wen, B.J. Nikolau, E.S. Wurtele [2000] Plant Physiol 122: 1057-1071). Embryos accumulated between 3- and 15-fold more transcripts per unit total RNA than young leaf tissue; the lower quantity of leaf 3-oxoacyl-ACP reductase mRNA was confirmed by reverse transcriptase-polymerase chain reaction. This is in conflict with analysis of B. napus transcripts using an Arabidopsis microarray (T. Girke, J. Todd, S. Ruuska, J. White, C. Benning, J. Ohlrogge [2000] Plant Physiol 124: 1570-1581) where similar leaf to seed levels of fatty acid synthase component mRNAs were reported.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed2002

Expression of glucocorticoid receptor alpha- and beta-isoforms in human cells and tissues.

Alternative splicing of the human glucocorticoid receptor (GR) primary transcript generates two protein isoforms: GR-alpha and GR-beta. We investigated the expression of both GR isoforms in healthy human cells and tissues. GR-alpha mRNA abundance (x10(6) cDNA copies/microg total RNA) was as follows: brain (3.83 +/- 0.80) > skeletal muscle > macrophages > lung > kidney > liver > heart > eosinophils > peripheral blood mononuclear cells (PBMCs) > nasal mucosa > neutrophils > colon (0.33 +/- 0.04). GR-beta mRNA was much less expressed than GR-alpha mRNA. Its abundance (x10(3) cDNA copies/microg total RNA) was as follows: eosinophils (1.55 +/- 0.58) > PBMCs > liver > or = skeletal muscle > kidney > macrophages > lung > neutrophils > brain > or = nasal mucosa > heart (0.15 +/- 0.08). GR-beta mRNA was not found in colon. While GR-alpha protein was detected in all cells and tissues, GR-beta was not detected in any specimen. Our results suggest that, in physiological conditions, the default splicing pathway is the one leading to GR-alpha. The alternative splicing event leading to GR-beta is minimally activated.

Animals↗

PubMed2003

Epidermal growth factor receptor mRNA in peripheral blood of patients with pancreatic, lung, and colon carcinomas detected by RT-PCR.

The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. An RT-PCR assay was used to detect EGFR mRNA in the blood of 43 patients with pancreatic, lung (NSCLC), colon, and renal carcinomas. After an initial serum tube was drawn to clear the line of detached epithelial cells, a 3 ml sample of EDTA-anticoagulated blood was collected from each cancer patient and healthy control. Total RNA was isolated from each sample and from cancer cell lines. After reverse transcription using specific priming, outer and nested primers for EGFR were employed for cDNA amplification. RNA integrity was confirmed with RT-PCR amplification using beta2-microglobulin primers. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized. The assay was validated using Southern blotting and was capable of detecting a lower limit of 100 fg of total RNA from the A431 cell line. EGFR-positive cells were detected in 3/10 (30%) non-small cell lung cancer patients, 2/11 (18%) pancreatic cancer patients, and 2/16 (12.5%) colon cancer patients, but in 0/6 (0%) patients with renal carcinoma (16% of solid tumor patients overall). All 23 healthy controls were negative. This study is the first to apply RT-PCR for the detection of EGFR mRNA in the peripheral blood of patients with pancreatic and renal carcinomas, and it lends further support for the use of EGFR mRNA as a marker of CTCs in the blood of patients with certain types of solid tumors.

Adenocarcinoma↗

PubMed1993

The sheep erythropoietin gene: molecular cloning and effect of hemorrhage on plasma erythropoietin and renal/liver messenger RNA in adult sheep.

Erythropoietin (Epo) production was studied in adult sheep. Nine ewes, body weight (BW) 39 +/- 1.3 kg, were hemorrhaged a volume of blood equivalent to 1.6% BW, and sampled at 0, 2, 4, 6, 24 h. Oxygen content (O2 CT) decreased by 2.7 +/- 0.6 ml/dl at 2 h. Plasma immunoreactive (IR) Epo was only significantly increased at 24 h, from 18.5 +/- 3.5 to 40 +/- 10.7 mU/ml (mean +/- SEM). A further 5 ewes were bled extensively (2793 +/- 82 ml) over 54 h, and killed for Epo mRNA determination. The O2 CT decreased from 12.3 +/- 1.6 to 4.1 +/- 0.6 ml/dl, and plasma Epo increased from 15 +/- 4 to 1675 +/- 287 mU/ml. The sequence of ovine Epo cDNA was derived from the kidney RNA of a severely bled sheep using reverse transcription/polymerase chain reaction (RT/PCR), and from an ovine Epo genomic clone. The cDNA encodes a peptide of 194 amino acids, including a 27 amino acid signal peptide. The deduced amino acid sequence of sheep Epo shows 82%, 78% and 80% homology with mature Epos of human, mouse and monkey, respectively. The gene structure resembles closely those of human and mouse, with 5 exons and 4 introns. The expression of the ovine Epo gene in tissues from normal and hemorrhaged sheep was analysed by a competitive RT/PCR method. Epo mRNA was difficult to detect in liver from normal sheep, but was detectable at 0.01-0.04 amol/microgram total RNA in kidney from normal sheep. In the kidneys of severely bled sheep, the Epo mRNA levels (per micrograms total RNA) increased 400-1500-fold compared to that of normal kidneys, and were approximately 60-fold greater than those in the livers of the hemorrhaged sheep.

Amino Acid Sequence↗

PubMed2002

Osteoblasts produce soluble factors that induce a gene expression pattern in non-metastatic prostate cancer cells, similar to that found in bone metastatic prostate cancer cells.

BACKGROUND: Progressive prostate cancer typically metastasizes to bone where prostate cancer cells gain an osteoblast-like phenotype and induce osteoblastic metastases through unknown mechanisms. To investigate the biology of prostate cancer skeletal metastases, we compared gene expression between the non-metastatic LNCaP cell line and its derivative cell line C4-2B that metastasizes to bone. METHODS: Total RNA from LNCaP and C4-2B cell lines was isolated and used to probe membrane-based gene arrays (Comparison 1). Additionally, LNCaP cells were incubated in the absence or presence of conditioned media (CM) from a human osteoblast-like cell line (HOBIT) and total RNA from these cells was used to probe gene arrays (Comparison 2). Differential expression of genes was confirmed by RT-PCR. RESULTS: Of the 1,176 genes screened, 35 were differentially expressed between LNCaP and C4-2B cells (Comparison 1). HOBIT-CM induced differential expression of 30 genes in LNCaP cells (Comparison 2). Interestingly, 19 genes that were differentially expressed in C4-2B vs. LNCaP also displayed a similar expression pattern in LNCaPs grown in HOBIT-CM. These genes are primarily involved in motility, metabolism, signal transduction, tumorigenesis, and apoptosis. CONCLUSIONS: These results suggest that osteoblasts produce soluble factors that contribute to the progression of prostate cancer skeletal metastases, including their transition to an osteoblast-like phenotype. Additionally, these data provide targets to explore for further investigations towards defining the biology of skeletal metastases.

Bone Neoplasms↗

PubMed2000

Differential expression of ras in organs and embryos of shrimp Penaeus monodon (Crustacea: Decapoda).

Total RNA from shrimp hepatopancreas of Penaeus monodon showed three prominent bands that react with the shrimp ras probe, a 239-bp product, of approximately 4.8 kb (R1), 3.1 kb (R2) and 1.3 kb (R3) on the northern blot. The R1 is the least abundant. Analyses of total RNA from gill and heart were similar to each other. The highest expression of Ras was observed in the gill, while a negligible signal was detected with the Ras probe in muscle. Ras expression is developmentally regulated in embryonic stages of shrimp. Messenger RNA levels of ras were increased from a minimum in the nauplius stage to a maximum in the post-larvae stage for R1 and R2. R3 showed a maximum at the protozoea stage. On the other hand, the activity of protein geranylgeranyltransferase I was increased significantly in the early nauplius stage. No correlative increase of prenylation activity by protein geranylgeranyltransferase I was observed with the transcription activity of ras.

Amino Acid Sequence↗

PubMed2005

The aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) genes are not expressed in the rat heart.

Aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) catalyze the production of aldosterone and corticosterone, respectively, in the rat adrenal cortex. Recently, there has been some debate as to whether these corticosteroids are also produced in the hearts of rodents and humans, possibly contributing to the development of hypertrophy and myocardial fibrosis. To investigate this, we have used our established, highly sensitive real-time quantitative RT-PCR method to measure CYP11B1 and CYP11B2 mRNA levels in adrenal and cardiac tissue from several rat models of cardiovascular pathology. We have also studied isolated adult rat ventricular myocytes treated with angiotensin II and ACTH. Total RNA was isolated from the adrenal and cardiac tissue of 1) male Wistar rats with heart failure induced by coronary artery ligation and sham-operated controls; 2) stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats as controls; 3) cyp1a1Ren-2 transgenic rats and Fischer controls; 4) isolated adult Sprague-Dawley ventricular myocytes incubated with 11-deoxycorticosterone (DOC), DOC plus angiotensin II, or DOC plus ACTH. Adrenal CYP11B2 expression was significantly increased in transgenic rats compared with Fischer controls (1.3 x 10(9)+/- 1.2 x 10(9) vs. 2.1 x 10(7) +/- 7.0 x 10(6) copies/microg RNA; P < 0.05). There were no other significant differences in adrenal CYP11B2 or CYP11B1 expression between the model animals and their respective controls. Cardiac CYP11B1 and CYP11B2 mRNA transcript levels from all in vivo and in vitro groups were never greater than 100 copies per microgram total RNA and therefore too low to be detected reproducibly. This suggests that cardiac corticosteroid production is unlikely to be of any physiological or pathological significance.

Adrenal Glands↗

PubMed1993

Analysis of proopiomelanocortin (POMC) messenger ribonucleic acid and POMC-derived peptides in human peripheral blood mononuclear cells: no evidence for a lymphocyte-derived POMC system.

A number of recent studies suggest that cells of the immune system, e.g. peripheral blood mononuclear cells (PBMC), can synthesize and process POMC and secrete POMC-derived peptides, such as ACTH and endorphins, upon immune and hormonal challenges. From this, it has been proposed that POMC-derived peptides originating from lymphoid cells can function as hormones, for instance in a lymphoid-adrenal axis. In view of the important physiological implications of this proposal, the present study was designed to investigate the expression of the POMC gene in human PBMC and the production by these cells of alpha-, beta-, and gamma-endorphins (alpha E, beta E, and gamma E) peptides that are established end products of the posttranslational processing of POMC. PBMC of individual donors were used uncultured (fresh cells) or cultured for 24 and 48 h in the presence and absence of Concanavalin-A (Con-A), bacterial lipopolysaccharide, phytohemagglutinin, or CRH, and vasopressin, conditions that reportedly stimulate POMC activity in those cells, to investigate the presence of POMC transcripts by analysis of total RNA with Northern blotting and the reverse transcriptase polymerase chain reaction (RT-PCR). Large scale preparations containing over 10(9) cells (fresh, cultured with and without Con-A) originating from several donors were examined for the presence of POMC transcripts by analysis of poly(A)+ RNA on Northern blots and for the presence of alpha E, beta E, and gamma E by gel filtration over Sephadex G-75 and reverse phase HPLC, followed by assay of the fractions in four endorphin RIA systems with different specificities. On the Northern blots of total RNA, no POMC transcripts were detectable. In poly(A)+ RNA preparations, no full-length POMC mRNA was found, and it was estimated that the concentration of POMC mRNA, if present, was below approximately 0.005 transcript/cell in Con-A-stimulated cells and still lower in unstimulated cells. In accord with literature data, an 800- to 900-nucleotide POMC transcript was detected in cultured PBMC, and the levels of this transcript were stimulated by Con-A. In all samples analyzed with RT-PCR, a transcript spanning most of exons 2 and 3 was detectable only on Southern blots of the RT-PCR product, but not on agarose gels stained with ethidium bromide. Chromatographic analysis of endorphin immunoreactivities in cell extracts revealed no qualitative differences between the immunoreactive profiles of fresh PBMC or PBMC cultured with or without Con-A.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed2002

Microsomal triglyceride transfer protein: does insulin resistance play a role in the regulation of chylomicron assembly?

We have previously demonstrated that diabetes is associated with an increase in intestinal microsomal triglyceride transfer protein (MTP) mRNA in both the rat and rabbit models. The present study was designed to investigate the relationship between MTP expression and chylomicron assembly in an insulin resistant non-diabetic animal model. Ten insulin resistant Zucker obese fa/fa rats and ten lean fa/minus sign rats were examined at 8-10 weeks of age. The lymph duct was cannulated and lymph collected for 4 h. Lymph chylomicrons were isolated by ultracentrifugation and their composition determined. RNA was extracted from intestinal mucosa and from the liver. MTP mRNA was measured using the RNase protection assay. Blood sugar in the fatty rats was significantly higher (6.3 +/-1.2 vs. 5.4 +/-0.4 P<0.05) and plasma insulin was almost six times that of the lean rats (P<0.001). Plasma cholesterol and phospholipid but not triglyceride were significantly increased in the obese animals (P<0.01). Obese animals secreted significantly more lymph chylomicron apo B48 (0.05 +/-0.02 vs. 0.02 +/-0.01 mg/h P<0.005), triglyceride (9.7 +/-5.3 vs. 3.8+/-1.9 mg/h P<0.005) and phospholipid (1.5 +/-0.7 vs. 0.4 +/-0.3 mg/h P<0.001). The only difference in the chylomicron particle composition between the two groups was a significant increase in phospholipid (P<0.01). Intestinal MTP mRNA expression was significantly higher in the fatty compared to the lean rats (22.1 +/-9.5 vs. 7.8+/-5.6 amol MTP mRNA/microg total RNA P<0.001) as was hepatic MTP mRNA expression (6.9 +/-3.5 vs. 3.4 +/-1.5 amol MTP mRNA/microg total RNA, P<0.01). Thus in this animal model of insulin resistance, increased MTP, which was associated with increased chylomicron particle number, may play a crucial role in the development of atherosclerosis.

Animals↗

PubMed2002

Induction of metallothionein-I protects glomeruli from superoxide-mediated increase in albumin permeability.

Metallothioneins (MT) are low-molecular-weight, heat-stable, cysteine-rich proteins with four isoforms. MT-I and MT-II are ubiquitous and are induced by oxidative, physical, and chemical stress. MT-I is an efficient scavenger of superoxide (*O2) and hydroxyl ion (OH(-)). We have demonstrated that *O2 and hypohalous acid can cause an increase in glomerular albumin permeability (P(alb)) in vitro. The purpose of this study was to document the protective effect of MT gene product on the *O2-mediated increase in P(alb). Glomeruli from Sprague-Dawley rats in 4% BSA medium were incubated for 4 hr at 37 degrees C in duplicate tubes. Each set contained glomeruli alone or with 5 microM Cd(++), 0.3 mM Spermine-NONOate (NO donor), 0.3 mM Sulfo-NONOate (nitrous oxide donor), 0.6 mM SNP (nonspecific NO donor) and SNP + carboxy-PTIO (10 mg/ml). After incubation, one set of tubes was used to isolate total RNA for the measurement of the mRNA levels of MT-I by reverse transcriptase polymerase chain reaction (RT-PCR). Duplicate tubes were incubated for an additional 10 min with 10 nM of *O2, and P(alb) was measured using video microscopy. RT-PCR of total RNA from Cd(++) and Spermine-NONOate treated glomeruli revealed a 2-fold induction of MT-I expression at the mRNA level. *O2 caused a significant increase in P(alb) (0.8 +/- 0.06 vs. control 0.0 +/- 0.12, P < 0.05) and induction of MT-I in glomeruli by Cd(++) or by Spermine-NONOate blocked this effect (0.21 +/- 0.12 and 0.24 +/- 0.19, respectively, P < 0.05 vs. *O2). In contrast, Sulfo-NONOate and SNP did not induce mRNA for MT-I in glomeruli and did not provide protection against *O2-mediated increase in P(alb.) We conclude that MT-I gene products may play an important role in protecting the glomerular filtration barrier from the injury induced by reactive oxygen species in immune and/or nonimmune renal diseases.

Animals↗

PubMed1988

Lack of expression of glutathione-S-transferase P, gamma-glutamyl transpeptidase, and alpha-fetoprotein messenger RNAs in liver tumors induced by peroxisome proliferators.

Many structurally unrelated nonmutagenic peroxisome proliferators induce altered areas, neoplastic nodules, and hepatocellular carcinomas in rats. Unlike the lesions induced by genotoxic hepatocarcinogens, these lesions do not stain positively for the phenotypic markers gamma-glutamyl transpeptidase (GGT) and glutathione-S-transferase P (GST-P). To ascertain whether the absence of immunocytochemically detectable GST-P and GGT proteins in peroxisome proliferator-induced neoplastic lesions is due to the absence of specific mRNAs, we analyzed the total RNA isolated from hepatocellular carcinomas induced by three different peroxisome proliferators (ciprofibrate, Wy-14643, and BR-931) and the genotoxic carcinogens, 2-acetylaminofluorene and aflatoxin B1 (AFB), for the presence of GST-P, GGT, and alpha-fetoprotein (AFP) mRNAs. Northern and dot blot analysis of total RNA isolated from liver tumors induced by three different peroxisome proliferators revealed no detectable GST-P, GGT, and AFP mRNAs. GST-P mRNA was also not detected in a transplantable hepatocellular carcinoma established from a liver tumor induced by ciprofibrate. In contrast, GST-P mRNA levels were high in primary liver tumors induced by both 2-acetylaminofluorene and AFB and the two transplantable hepatocellular carcinomas established from such tumors. By immunoblot method, GST-P protein was found to be abundant in both primary and transplantable liver tumors induced by genotoxic carcinogens but not in those derived from peroxisome proliferator treatment. The GGT and AFP mRNAs were also not found in all 18 liver tumors induced by peroxisome proliferators that were analyzed and also in the ciprofibrate-derived transplantable liver tumor. The expression of GGT and AFP genes in liver tumors induced by 2-acetylaminofluorene and AFB was variable. These studies with peroxisome proliferators show that the GST-P and GGT gene derepression is not essential for the hepatocarcinogenesis or successful tumor transplantation. Further characterization of the molecular basis for the differential expression, particularly of the GST-P gene in liver tumors, may help identification of the critical event(s) in hepatocarcinogenesis by genotoxic carcinogens and nongenotoxic peroxisome proliferators.

Animals↗

PubMed1985

Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells). Dissociation between nuclear iodothyronine receptor concentration and growth hormone mRNA synthesis during the deoxyribonucleic acid synthesis phase of the cell cycle.

3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labeling with [3H]leucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehyde-treated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p less than 0.005 (from 13,430 +/- 609 to 19,150 +/- 1160 cpm/10(6) cells/2 h) and increased GH mRNA, p less than 0.001 (from 7.2 +/- 1.2 to 14.5 +/- 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cell populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/10(6) cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗

PubMed1998

Pattern of messenger ribonucleic acid expression of tissue inhibitors of metalloproteinases (TIMPs) during testicular maturation in male mice lacking a functional TIMP-1 gene.

It has been proposed that proteolytic remodeling of the testicular extracellular matrix (ECM) plays a fundamental role in testicular development, morphogenesis, and spermatogenesis. Tissue inhibitor of metalloproteinase (TIMP)-1 regulates ECM turnover and has been reported to stimulate Leydig cell steroidogenesis. To assess the developmental changes in TIMP mRNA expression and the potential steroidogenic role of TIMP-1 in testicular physiology, an experiment was conducted that used male mice incapable of expressing the TIMP-1 gene product. TIMP-1-deficient and wild-type male mice (n = 6 to 15 per age group per genotype) were killed at 18, 21, 24, 27, 33, 41, and 49 days of age. Body weight, testis weight, serum total testosterone, and TIMP-1, -2, -3, and -4 transcript expression were determined. Northern analysis revealed the detection of TIMP-1 mRNA in wild-type males only. TIMP-1 mRNA levels (per 20 microg total RNA) were highest in 18- to 27-day-old male mice and decreased approximately 13-fold by Day 41. The pattern of TIMP-2 expression was similar between genotypes, with testicular levels of the 1. 0-kilobase transcript increasing between Days 18 and 27 of age. The pattern of TIMP-3 transcript expression (per 20 microg total RNA) was similar between genotypes and decreased between Days 18 and 41 of age. When TIMP-3 mRNA levels were expressed on a per testis basis, TIMP-3 was seen to have increased throughout testicular development. TIMP-4 mRNA expression was undetectable by Northern analysis in all mice. No significant difference was detected in body weight or testis weight between genotypes, with the exceptions that 21-day-old TIMP-1 mutants had higher (p < 0.05) testis weights and lower (p < 0. 05) serum total testosterone levels than age-matched wild-type males. It is concluded that each TIMP displays its own unique pattern of expression during the prepubertal period, suggesting that the various TIMPs may have specific roles in testicular development. The modest effect of TIMP-1 ablation on testosterone is interpreted to mean that TIMP-1 may function as a coregulator of basal testicular steroidogenesis; but overall, TIMP-1 appears to have little effect on testosterone production in mice lacking the TIMP-1 gene.

Aging↗

PubMed1985

Increase of globin RNA synthesis induced by phosphatidylserine liposomes in isolated erythroleukemic cell nuclei. Morphological and functional features.

The effect of phospholipid vesicles on chromatin structure, protein composition and globin RNA synthesis has been analysed in nuclei isolated from murine erythroleukemia cells. In terms of chromatin organisation, PC vesicles with neutral surface charge do not affect the structure of chromatin fibres, whereas negatively charged PS vesicles induce chromatin decondensation to a great extent. Indeed the fibres appear uniformly dispersed lacking also the perinucleolar heterochromatin. These morphological features are accompanied by depletion of lysine-rich histones H1 and H1(0) and of histone-like protein A5, due to PS liposomes. Functionally, PS vesicles induce enhancement both of total RNA and specific mRNA synthesis, as analysed by in vitro transcription of beta globin gene. On the contrary, PC vesicles do not seem to affect significantly total RNA and globin mRNA synthesis. These observations fit well with previous data obtained in other experimental systems, and support both the use of these molecules as probes for chromatin structure and function and their possible involvement in transcriptional events.

Amanitins↗

PubMed2002

Isolation of small-subunit rRNA for stable isotopic characterization.

Small-subunit ribosomal RNA (SSU rRNA) has several characteristics making it a good candidate biomarker compound: it is found in bacteria, archaea and eukaryotes; it is quickly degraded extracellularly, hence SSU rRNA extracted from a sample probably derives from the currently active population; it includes both conserved and variable regions, allowing the design of capture probes at various levels of phylogenetic discrimination; and rRNA sequences from uncultured species can be classified by comparison with the large and growing public database. Here we present a method for isolation of specific classes of rRNAs from mixtures of total RNA, employing biotin-labelled oligonucleotide probes and streptavidin-coated paramagnetic beads. We also show that the stable carbon isotope composition of Escherichia coli total RNA and SSU rRNA reflects that of the growth substrate for cells grown on LB, M9 glucose and M9 acetate media. SSU rRNA is therefore a promising biomarker for following the flow of carbon, and potentially nitrogen, in natural microbial populations. Some possible applications are discussed.

Carbon Isotopes↗

PubMed1997

Insulin-like growth factor I receptor messenger RNA in the colon is unchanged during neoplasia.

The expression of growth factor receptor messenger RNA is difficult to quantitate due to low copy number. We describe a quantitative polymerase chain reaction that rapidly, reproducibly assays expression of human insulin-like growth factor I (IGF-I) receptor mRNA from small, biopsy-sized, specimens of tissue. This was then clinically applied to surgically resected specimens of colon. Total RNA was isolated from normal colonic mucosa and documented tumors from 4 patients undergoing resection. The mRNA was first reverse-transcribed with an oligomer bearing a complementary sequence specific for the mRNA at its 3' end, and a sequence complementary to an intervening intron of the IGF-I receptor gene at the 5' end. Competitive PCR was then performed in the presence of the cDNA product and exogenously added genomic DNA, with an upstream primer complementary to the exon sequence of the gene of interest and a downstream primer complementary to the intron sequence that was tagged to the cDNA. The genomic DNA was used as the internal standard. To calculate the number of copies of mRNA per microgram total RNA, a standard curve was used. No difference was noted in IGF-I receptor expression between neoplastic and normal colonic mucosa. This quantitative PCR is accurate, rapid, and requires very small amounts of tissue. Potential uses are in determining genetic expression of growth factor receptors or putative tumor markers preoperatively from small samples obtained during diagnostic colonoscopy or from samples obtained at surgery.

Colon↗